The long QT syndrome (LQTS) is a disorder associated with delayed cardiac repolarization, prolonged electrocardiographic QT intervals, and the development of ventricular arrhythmias (torsades de pointes) and sudden death. LQTS can be congenital (inherited), and one of the principal causes of congenital LQTS are mutations in the human ether-a-go-go-related gene (HERG) producing chromosome 7-linked congenital LQTS or LQT2. HERG encodes the pore-forming subunit of a voltage-gated potassium channel. HERG channel current has properties similar to the rapidly activating delayed rectifier potassium current (IKr) which plays a key role in cardiac action potential repolarization in the mammalian heart. In addition to congenital LQTS, HERG channels are important targets for blockade by many drugs, and it is well established that drug-induced suppression of HERG current causes action potential prolongation and cardiac arrhythmias. This has led to the withdrawal from the world market of several prescription drugs including terfenadine (Seldane), astemizole (Hismanal), cisapride (Propulsid), as well as restriction in the use of other drugs or their failure during drug development because of potential QT intervalrelated toxicity. Therefore, HERG channels have emerged as a very important cardiac ion channel.
A focus of our laboratory has been the, study of HERG potassium channels and how they are involved in the congenital (inherited) and acquired (drug-induced) LQTS. In these studies we use isolated native mammalian heart cells along with cells derived from human origin (human embryonic kidney 293 or HEK293 cells) that are transfected with HERG cDNA encoding the normal (wild-type, see Zhou, et al., Biophys. J. 74:230-241, 1998) gene or encoding mutated genes usually of a known human LQT2 mutation (see Zhou, et al., J. Biol. Chem. 273:21061-21066, 1998; Furutani, et al., Circulation 99:2290-2294, 1999). Cells are then studied using patch clamp electrophysiological, biochemical and immunohistochemical methods to investigate the molecular mechanisms of HERG channel dysfunction caused by LQT2 mutations, and how wild-type and mutant channels are affected by drugs.
An important step in understanding the mechanisms of HERG channel dysfunction in LQT2 was the recognition that some mutations caused defects in biosynthetic processing of HERG channels with the channel protein retained intracellularly in the endoplasmic reticulum (e.g., the channel protein can not reach the cell surface membrane). Many mutations appear to work by this non-trafficking mechanism. However, some mutations are processed similarly to wild-type HERG protein but do not produce functional channels (e.g., channel protein reaches the cell surface membrane but does not work) and other mutations express HERG current but with altered gating properties (e.g., channel protein reaches the cell surface membrane but functions abnormally). These findings were presented in Zhou, et al (Zhou, et al., supra, 1998) and suggested that the loss of HERG channel function in LQT2 mutations is caused by multiple mechanisms including abnormal channel protein trafficking, the generation of nonfunctional channels, and altered channel gating.
We then attempted to “rescue” non-trafficking LQT2 mutations. It was known for a few human diseases that maintaining cells at low temperature rescued some non-trafficking disease-causing protein mutations. This had been shown in 1992 for some mutations in the CFTR channel in cystic fibrosis. We showed temperature correction of the trafficking defect for the LQT2 mutation N470D (asparagine to aspartate at amino acid position 470, see Zhou, et al., J. Biol. Chem. 274:31123, 1999). We had previously suggested this mechanism for the G601S (glycine to serine) LQT2 mutation (Furutani, et al., supra, 1999). When expressed at room temperature in Xenopus oocytes, these mutants generate functional HERG channels and in our HEK293 cells stably transfected with these LQT2 mutant channels, culturing the cells at 27° C. also results in the functional expression of HERG current. In the same cells, culturing at 37° C. results in very little current. Biochemical studies confirmed that the trafficking of the mutant HERG protein was temperature-dependent with the mutant protein retained intracellularly at physiological temperature. Although some LQT2 channels can be shown to have both functional and trafficking abnormalities, under physiological conditions abnormal trafficking appears to be the dominant defect.
In addition to lowering temperature, the trafficking defect for the N470D mutant channel can be rescued by pharmacological approaches (see Zhou, Gong, and January, supra, 1999; January, et al., J. Cardiovasc. Electrophysiol. 12:1413-1418, 2000). N470D mutant expressing HEK293 cells were cultured at physiological temperature in the presence of low concentrations of drugs known to block with high affinity HERG channels (the antiarrhythmic E-4031, the antihistamine astemizole, and the GI prokinetic agent cisapride). This resulted in the appearance on western blot analysis of the mature HERG protein band. When the drugs were washed off the cells large amplitude HERG current could be recorded confirming pharmacological rescue. Drugs that do not block HERG channels, such as nifedipine, do not mimic this effect. One interpretation of these findings is that drugs binding with high affinity to HERG channels may act as chemical or pharmacological chaperones to stabilize protein folding or assembly of mutant protein in a conformation that permits trafficking to the plasma membrane.